1 Background

In this report, we will focus on studying the distances, Delta MES and MSR results of the splicing noise analysis of Ataxia RNAseq samples.

In this report, we will focus on studying the results of the mis-splicing noise analysis of Ataxia RNAseq samples: 46 Cerebellum and 45 Frontal Cortex samples. Samples will be pseudobulked by tissue, status disease and ataxia diagnosis, splitting the analysis in three different levels, including the results of the splicing noise between specific Ataxia diagnosis (i.e. SCA1, FRDA…) and control samples.

Changelog

v2.1

  • Changed the reference transcriptome to Gencode v38.

v2.0

  • Now, the clusters always compare the same number of samples between cases and controls. Procedure explained in methodology.

  • Added results for Level 2 (AtaxiaSubtype) and references.

  • Changed the reference transcriptome from Gencode v43 to Gencode v39.

  • Updated methodology.

v1.1

  • Updated subsampling method to consider other covariates other than RIN. Each covariate was weighted to contribute according to its influence to the number of novel junctions for each sample. For both tissues, RIN was the most relevant contributor with more than 85% of the weight. Thus, no relevant variation from the previous approach was observed.

2 Methods

2.1 Analysis pipeline (splicing noise evaluation)

For every available BAM file to study, we apply the following steps:

  1. Download and extraction of BAM files: the files are downloaded from s3://ataxia-bulk-rnaseq/nextflow_first_attemp/Star_2_pass_by_indv/output/STAR/align/BAM_files/ and subfolders.

  2. Junction extraction: all junctions are extracted using regtools junction extract after sorting and indexing with samtools. A file is created for each BAM file in BED12 format.

  3. Junction annotation: the junctions are read from the previously created files and merged into a single dataframe of read junctions. We also register the number of reads of each junction in every sample. The junctions located within the ENCODE blacklisted regions v2 are removed. The junction_annot() function from the package dasper is used to annotate the junctions to the Gencode v38 reference transcriptome. All junctions not classified as either novel_donor, novel_acceptor or annotated are ignored. We also remove all junctions smaller than 25bp (base pairs) and annotated introns that are ambiguously assigned to more than one gene.

  4. Junction pairing: by looking for overlaps between the novel junctions and the annotated junctions for each sample, we measure the distance in bp between the novel and reference splice site. The annotated introns that are never associated to a novel junction are considered a never misspliced junctions.

  5. Filtering the distances: we remove the pairings in which a novel junctions are associated to more than one reference intron across different samples. For more information about this process, please see the methods section in Introverse paper [1].

Next, we need to decide on a clustering method to combine and compare different samples. More information in section about clustering.

  1. Measuring the mis-splicing ratio: by adding all novel junction read counts attached to an annotated intron across all samples in which the novel splice was observed, and then dividing by the total number of reads of the annotated intron and the novel junctions across the same set of samples, we obtain a measurement of the mis-splicing ratio for an given annotated intron at both the donor splice site and the acceptor splice site. For more information about the mis-splicing ratio, please see section MSR.

  2. Generation of the DB: two tables are created per each cluster: db_introns and db_novel. Each one contains the relevant information related to reference introns (including the never misspliced introns) and novel junctions. This includes the MaxEntScan scores, the percentage of protein-coding transcripts and the classification in u2 and u12 introns.

2.2 Clustering (pseudobulk)

In our dataset, we have a total of 95 samples, corresponding to 48 different individuals. A total of 4 samples are removed because they belong to individuals diagnosed as CANVAS and AIFM1.

Three different level of studies were studied in this report, and always different analyses for each tissue.

  • Level 1 (Type): whether the sample is diagnosed with ataxia or not (diases status). For the frontal cortex tissue, samples with \(RIN<4\) will be removed, while for the Cerebellum tissue only controls with \(RIN<=7\) are kept. This is to ensure non-significant differences in the RIN medians.

  • Level 2 (AtaxiaSubtype): two different analyses are performed: known ataxia cases vs. controls and unknown ataxia cases vs. controls. In both scenarios, no restrictions about RIN is required.

  • Level 3 (Diagnosis): a different analysis was performed for every ataxia diagnosis with at least three samples: FRDA, SCA1, SCA2 and SCA6. In all situations, control samples are selected to minimize a weighted Gower distance to the case samples (more information in following section).

2.3 Subsampling

Studies about the relationship in mis-splicing ratio’s median and the number of samples pseudobulked showed a clear correlation between the two. In order to avoid this effect in the comparisons between cases and controls, we decided to subsample the majority class until both classes have the same number of samples. The subsampling was performed so that the weighted Gower distance between the case samples and the control samples was minimized.

For datasets with both quantitative (i.e. RIN) and categorical (i.e. Brain bank) variables, also called mixed datasets, Gower’s distance is a common measurement of similarity between any two samples [2]. The similarity between samples can be defined as:

\[ S_{ij}=\frac{\sum_{k=1}^p s_{ijk}\delta_{ijk}w_k}{\sum_{k=1}^p \delta_{ijk}w_k} \] where \(p\) are the variables beeing compared, \(i\) and \(j\) refers to two different samples and:

  • Quantitative variables: \(s_{ijk}=1-|x_{ik}-x_{jk}|/R_k\) where \(R_k\) is the range of the variable \(k\).
  • Categorical variables: \(s_{ijk}=1\) if \(x_{ik}=x_{jk}\) and \(s_{ijk}=0\) otherwise.
  • \(\delta_{ijk}\): whether a comparison between sample \(i\) and sample \(j\) can be performed for variable \(k\).
  • \(w_k\): optional weight to increase or decrease the relevance of certain variables.

We decided to apply a weighting to the variables in order to increase the relevance of the main contributors to the number of junction reads across samples.

The subsampling process was the following:

  1. Variable weights: first, we divided the samples by tissue. Then, for each sample, we extract the number of reads associated to both annotated and novel junctions. Using a linear model to predict this number of reads, we measure the variance explained by each of the main covariates that will be employed in subsampling: RIN, PMI, Age at death, Brain bank and sex. The percentage of variance explained by each covariate will be considered as the weight in the next step.

  2. Gower’s distance between samples: for each the minority class sample, the most similar majority class sample was selected without repetition (i.e. once two samples were assigned together, none of them can be selected again). Thus, the same number of samples between the two classes were obtained.

  3. Wilcoxon test: between the two sets of samples, a Wilcoxon test is executed to test whether there are significant differences in the RIN medians. If significant differences are found, some extra restriction is applied to any of the sets. For example, for Cerebellum Level 1 study, control samples with RIN higher than 7 needed to be removed to ensure non-significant differences in the median.

The obtained weights are the following:

Covariate Cerebellum weights Frontal Cortex weights
RIN 0.989 0.855
PMI 0.001 0.132
Brain Bank 0.003 0.013
Age at death 0.001 0.000
Sex 0.006 0.000

2.4 Common annotated introns

For each study, we will only consider the common annotated introns between the two classes. To do so, we generate the following dataframes:

  • Common annotated intron table: we looped through both db_introns tables and extracted only the information from the common annotated introns in the clusters. To identify common annotated introns, we used their locus (i.e. seqname:start-end:strand), since it is a unique identifier. The goal is to have the same number of annotated between cases and controls.

  • Common novel junction table: we looped through both db_novel tables and extracted only the information from the novel junctions associated to common annotated introns. Thus, we first needed to calculate the common annotated intron table.

3 Studied metrics

The examples in this section corresponds to the Level 1 study for Cerebellum.

Distances

The distances graph is generated by counting the number of unique novel junctions at any given distance of the reference splicing site. From the list of novel junctions associated to common reference introns, we group by novel_type and cluster, count the number of entries for each distance within 30 bp into both intron and exon sequence, and represent the data in a histogram. An example of the data can be seen in the following table:

Novel jun. ID Novel type Cluster Sequence Distance [bp]
1900408 novel_acceptor Control intron -26
262172 novel_acceptor Case exon 4
1562780 novel_acceptor Case exon 75
1744154 novel_acceptor Control exon 26
2006682 novel_acceptor Case exon 84
315218 novel_donor Control exon 3
1123582 novel_acceptor Control intron -22
149464 novel_acceptor Case exon 89
951814 novel_donor Case exon 10
446245 novel_acceptor Case exon 29

For each study, different graphical representations will be presented. First, we present a histogram of the different distance values between -30 and 30 bp, with the case bars stacked on top of the control bars:

However, this representation does not allow for an easy comparison between cases and controls because of the bar stacking. To do so, we split the graph in two more facets to represent each cluster by its own:

If we focus on the difference in the number of unique novel junctions between cases and controls, the last graph represent the histogram resulting from subtracting the control distances from the case distances:

Distances (modulo 3)

To generate the modulo 3 graphs, we first filter by those distances smaller than 100 bp in either direction. Then, we group by novel_type and cluster, and calculate the distance in modulo 3 (i.e. the remainder from the division of the distance by 3). Once we have all novel junctions split in the four different categories with their distances in modulo 3, we calculate the percentage that each modulo 3 represents in each category. Thus, for each category:

\[ \text{mod.0 }\% = \frac{\# 0}{\#0 + \#1 + \#2}*100\%\qquad\quad \text{mod.1 }\% = \frac{\# 1}{\#0 + \#1 + \#2}*100\%\qquad\quad \text{mod.2 }\% = \frac{\# 2}{\#0 + \#1 + \#2}*100\% \]

Here, \(\#X\) represents the number of novel junctions at a modulo 3 distance equal to \(X\). For example, from the information of the following table, we can focus on the Case acceptor group, where we observe two 0s, two 1s and one 2:

Novel jun. ID Novel type Cluster Sequence Distance [bp] Mod3 distance [bp]
Case acceptor
262172 novel_acceptor Case exon 4 1
1562780 novel_acceptor Case exon 75 0
2006682 novel_acceptor Case exon 84 0
149464 novel_acceptor Case exon 89 2
446245 novel_acceptor Case exon 29 2
Case donor
951814 novel_donor Case exon 10 1
Control acceptor
1900408 novel_acceptor Control intron -26 2
1744154 novel_acceptor Control exon 26 2
1123582 novel_acceptor Control intron -22 1
Control donor
315218 novel_donor Control exon 3 0

Thus, the calculated percentage are:

\[ \text{mod.0 }\% = 40\%\qquad\quad \text{mod.1 }\% = 40\%\qquad\quad \text{mod.2 }\% = 20\% \]

Additionally, we can further group the novel junctions by their intron/exon sequence, resulting a total of 8 categories, where the same calculation of percentages can be applied:

Novel jun. ID Novel type Cluster Sequence Distance [bp] Mod3 distance [bp]
Case acceptor exon
262172 novel_acceptor Case exon 4 1
1562780 novel_acceptor Case exon 75 0
2006682 novel_acceptor Case exon 84 0
149464 novel_acceptor Case exon 89 2
446245 novel_acceptor Case exon 29 2
Case donor exon
951814 novel_donor Case exon 10 1
Control acceptor exon
1744154 novel_acceptor Control exon 26 2
Control acceptor intron
1900408 novel_acceptor Control intron -26 2
1123582 novel_acceptor Control intron -22 1
Control donor exon
315218 novel_donor Control exon 3 0

With the previous tables in mind, we represent them in three different ways. The first one just represents the number of unique novel junctions for each modulo 3 distance where \(abs(distance) <= 100\).

We also represent the percentage that each that each distance in modulo 3 represents when grouped by novel_type (i.e. acceptor/donor) and cluster (i.e. case/control).

Lastly, we represent the same data as before but taking into consideration whether it is located in the intronic or exonic sequence:

Delta MaxEntScan Score

To represent the difference in MaxEntScan scores (MES), we subtract the MaxEntScan score of the annotated intron to the MaxEntScan score of a novel junction at each splice site. We obtain a table like the following, and represent the density plot of the different values for delta_ss5score and delta_ss3score:

\[ \text{Delta MES}^{5'} = \text{MES}_{ref}^{5'}-\text{MES}_{novel}^{5'}\qquad\qquad\text{Delta MES}^{3'} = \text{MES}_{ref}^{3'}-\text{MES}_{novel}^{3'} \]

Example table with the Delta MES data:

Novel jun. ID Ref. jun. ID Delta ss5score Delta ss3score
2035214 2035210 2.63 3.96
89328 89327 6.31 -0.30
67502 67507 3.53 -0.88
381357 381360 -2.17 0.00
1547859 1547861 2.25 1.54
1151075 1151074 -0.76 0.00
1877181 1877182 22.15 22.18
704572 704556 4.77 11.05
1533950 1533949 2.09 1.97
806079 806085 0.05 0.00

As for the visual representations, the difference between the MaxEntScan scores for each splice site is shown in the X-axis. The Y-axis represents the kernel density estimate (smoothed version of a histogram). The fill color represents cases vs. controls.

Additionally, we can also represent the difference in the kernel density estimate between case and control:

MSR

The Mis-Splicing Ratio (MSR) is calculated in the data-analysis pipeline for each annotated intron found in the project samples. More information about the mis-splicing ratio can be found in the Ataxia RNAseq Pseudobulk - Median MSR Analaysis report. To visualize this data, we represent the density plot for MSR at the different splice sites (i.e. donor or acceptor). Example table with the MSR data:

Ref. jun, ID Cluster Ref. type MSR Donor MSR Acceptor
1331421 Control never 0.000 0.000
1972189 Control both 0.059 0.004
414325 Case acceptor 0.000 0.033
750187 Control never 0.000 0.000
1996411 Case never 0.000 0.000
573131 Case donor 0.004 0.000
253464 Case never 0.000 0.000
448971 Control never 0.000 0.000
403929 Case never 0.000 0.000
1503717 Control never 0.000 0.000

We represent both the distribution of mis-splicing ratio (MSR) at the donor site and the acceptor site. The X-axis represents the MSR, while the Y-axis represents the kernel density estimate.

## <br><br><br>

Note that the distributions have a discontinuity at \(x=0.05\) to better represent all possible MSR values. In some cases, a discontinuity in the Y-axis will also be present if the difference between the two sample types exceeds a predefined threshold.

Stats

Under the Stats subsection, we will study different statistics of the annotated introns and novel junctions:

  • Unique annotated introns: number of annotated introns classified as never mis-spliced, mis-spliced at acceptor end, donor end or both. It is represented by cluster, so that we can study the variation in the number and percentage of never mis-spliced annotated introns.

  • Reads - annotated introns: total reads associated to annotated introns and the percentage that they represent against the total read depth.

  • Reads - novel junctions: total reads associated to novel junctions and the percentage that they represent against the total read depth. Results are separated by cluster.

4 Results for Level 1 (Type)

4.1 Cerebellum

Distribution of sample RIN for Cerebellum level 1 study:

Distances (N)

Expand details
  • X-Axis: Distance between the novel splice site and the reference splice site.
  • Y-Axis: Number of unique novel junctions. Bars are stacked.
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details
  • X-Axis: Distance between the novel splice site and the reference splice site.
  • Y-Axis: Number of unique novel junctions.
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor) and cluster (same as colour).

Expand details
  • X-Axis: Distance between the novel splice site and the reference splice site.
  • Y-Axis: Difference between the number of unique novel junctions from case samples and from control samples.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor).

Modulo 3

Expand details
  • X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
  • Y-Axis: Number of unique novel junctions. Also represented as the text inside the bars.
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details
  • X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
  • Y-Axis: Percentage of each modulo 3 value with respect to cluster (case and control) and novel type (donor and acceptor).
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details
  • X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
  • Y-Axis: Percentage of each modulo 3 value with respect to cluster (case and control), novel type (donor and acceptor) and sequence (intronic and exonic).
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor) and sequence (intronic vs. exonic).

Delta MaxEntScan

Expand details
  • X-Axis: Difference between the MaxEntScan score of the reference annotated intron and the novel junction.
  • Y-Axis: Kernel density estimate (smoothed version of a histogram).
  • Colour: Cluster type as case vs. control.
  • Facets: Different splice sites.

Expand details
  • X-Axis: Difference between the MaxEntScan score of the reference annotated intron and the novel junction.
  • Y-Axis: Difference between the kernel density estimate for case samples and control samples.
  • Colour: Cluster type as case vs. control.
  • Facets: Different splice sites.

MSR

## <br><br><br>

Expand details
  • X-Axis: The mis-splicing ratio.
  • Y-Axis: Kernel density estimate.
  • Colour: Cluster type as case vs. control.
  • Facets: Different splice sites.
  • Notes: Distributions have a discontinuity at X = 0.05 to better represent all possible MSR values. In some cases, a discontinuity in the Y-axis is also present if the difference between the two sample types exceeds a predefined threshold.

Stats

Unique annotated introns

Mis-spliced site Cluster # Annotated introns # Ann. introns by splice site Percent [%]
acceptor Case 198167 40435 20.40
acceptor Control 198167 39710 20.04
both Case 198167 33845 17.08
both Control 198167 34144 17.23
donor Case 198167 21951 11.08
donor Control 198167 21934 11.07
never Case 198167 101936 51.44
never Control 198167 102379 51.66
Expand details
  • # Annotated introns: number of total annotated introns in the cluster. Since we limit the annotated introns to those common across all samples and clusters, the number is the same for both clusters.
  • # Ann. introns by splice site: the number of annotated introns for the cluster and splice site.
  • Percent [%]: the percentage that the annotated introns in the splice site represent to the total number of introns in the cluster:
\[ \text{Percent [%]}=\frac{\text{# Ann. introns by splice site}}{\text{# Annotated introns}}*100\% \]

Reads - annotated introns

Cluster # Annotated introns # Reads in cluster # Ann. intron reads Percentage [%]
Case 198167 4052024612 423466847 10.45
Control 198167 4520672127 521789300 11.54
Expand details
  • # Annotated introns: number of common annotated introns in the cluster. Since we limit the annotated introns to those common across all samples and clusters, the number is the same for both clusters.
  • # Reads in cluster: total number of reads from the BAM files that constitutes the cluster.
  • # Ann. intron reads: reads associated to the annotated introns in the cluster. Only common introns are being counted.
  • Percent [%]: the percentage that the annotated intron reads represent to the total number of reads in the cluster.
\[ \text{Percent [%]}=\frac{\text{# Ann. intron reads}}{\text{# Reads in cluster}}*100\% \]

Reads - novel junctions

Cluster Novel type # Novel junctions # Novel junc. in splice site # Reads in cluster # Novel junc. reads Percentage [%]
Case novel_acceptor 234748 136989 4052024612 2355486 0.06
Case novel_donor 234748 97759 4052024612 1700082 0.04
Control novel_acceptor 238281 137983 4520672127 2803847 0.06
Control novel_donor 238281 100298 4520672127 2003931 0.04
Expand details
  • # Novel junctions: total number of novel junctions for that particular cluster.
  • # Novel junc. in splice site: total number of novel junctions for that particular splice site in the cluster.
  • # Reads in cluster: total number of reads from the BAM files that constitutes the cluster.
  • # Novel junc. reads: reads associated to the novel junctions in the cluster and splice site.
  • Percent [%]: the percentage that the novel junction reads represent to the total number of reads in the cluster.
\[ \text{Percent [%]}=\frac{\text{# Novel junc. reads}}{\text{# Reads in cluster}}*100\% \]

4.2 Frontal Cortex

Distribution of sample RIN for Frontal Cortex level 1 study:

Distances (N)

Expand details
  • X-Axis: Distance between the novel splice site and the reference splice site.
  • Y-Axis: Number of unique novel junctions. Bars are stacked.
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details
  • X-Axis: Distance between the novel splice site and the reference splice site.
  • Y-Axis: Number of unique novel junctions.
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor) and cluster (same as colour).

Expand details
  • X-Axis: Distance between the novel splice site and the reference splice site.
  • Y-Axis: Difference between the number of unique novel junctions from case samples and from control samples.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor).

Modulo 3

Expand details
  • X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
  • Y-Axis: Number of unique novel junctions. Also represented as the text inside the bars.
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details
  • X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
  • Y-Axis: Percentage of each modulo 3 value with respect to cluster (case and control) and novel type (donor and acceptor).
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details
  • X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
  • Y-Axis: Percentage of each modulo 3 value with respect to cluster (case and control), novel type (donor and acceptor) and sequence (intronic and exonic).
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor) and sequence (intronic vs. exonic).

Delta MaxEntScan

Expand details
  • X-Axis: Difference between the MaxEntScan score of the reference annotated intron and the novel junction.
  • Y-Axis: Kernel density estimate (smoothed version of a histogram).
  • Colour: Cluster type as case vs. control.
  • Facets: Different splice sites.

Expand details
  • X-Axis: Difference between the MaxEntScan score of the reference annotated intron and the novel junction.
  • Y-Axis: Difference between the kernel density estimate for case samples and control samples.
  • Colour: Cluster type as case vs. control.
  • Facets: Different splice sites.

MSR

## <br><br><br>

Expand details
  • X-Axis: The mis-splicing ratio.
  • Y-Axis: Kernel density estimate.
  • Colour: Cluster type as case vs. control.
  • Facets: Different splice sites.
  • Notes: Distributions have a discontinuity at X = 0.05 to better represent all possible MSR values. In some cases, a discontinuity in the Y-axis is also present if the difference between the two sample types exceeds a predefined threshold.

Stats

Unique annotated introns

Mis-spliced site Cluster # Annotated introns # Ann. introns by splice site Percent [%]
acceptor Case 212490 42989 20.23
acceptor Control 212490 42227 19.87
both Case 212490 38202 17.98
both Control 212490 37676 17.73
donor Case 212490 25739 12.11
donor Control 212490 25323 11.92
never Case 212490 105560 49.68
never Control 212490 107264 50.48
Expand details
  • # Annotated introns: number of total annotated introns in the cluster. Since we limit the annotated introns to those common across all samples and clusters, the number is the same for both clusters.
  • # Ann. introns by splice site: the number of annotated introns for the cluster and splice site.
  • Percent [%]: the percentage that the annotated introns in the splice site represent to the total number of introns in the cluster:
\[ \text{Percent [%]}=\frac{\text{# Ann. introns by splice site}}{\text{# Annotated introns}}*100\% \]

Reads - annotated introns

Cluster # Annotated introns # Reads in cluster # Ann. intron reads Percentage [%]
Case 212490 4769291070 854565112 17.92
Control 212490 5300507311 862005120 16.26
Expand details
  • # Annotated introns: number of common annotated introns in the cluster. Since we limit the annotated introns to those common across all samples and clusters, the number is the same for both clusters.
  • # Reads in cluster: total number of reads from the BAM files that constitutes the cluster.
  • # Ann. intron reads: reads associated to the annotated introns in the cluster. Only common introns are being counted.
  • Percent [%]: the percentage that the annotated intron reads represent to the total number of reads in the cluster.
\[ \text{Percent [%]}=\frac{\text{# Ann. intron reads}}{\text{# Reads in cluster}}*100\% \]

Reads - novel junctions

Cluster Novel type # Novel junctions # Novel junc. in splice site # Reads in cluster # Novel junc. reads Percentage [%]
Case novel_acceptor 270355 152012 4769291070 2948776 0.06
Case novel_donor 270355 118343 4769291070 2176172 0.05
Control novel_acceptor 265070 147966 5300507311 3030018 0.06
Control novel_donor 265070 117104 5300507311 2263035 0.04
Expand details
  • # Novel junctions: total number of novel junctions for that particular cluster.
  • # Novel junc. in splice site: total number of novel junctions for that particular splice site in the cluster.
  • # Reads in cluster: total number of reads from the BAM files that constitutes the cluster.
  • # Novel junc. reads: reads associated to the novel junctions in the cluster and splice site.
  • Percent [%]: the percentage that the novel junction reads represent to the total number of reads in the cluster.
\[ \text{Percent [%]}=\frac{\text{# Novel junc. reads}}{\text{# Reads in cluster}}*100\% \]

5 Results for Level 2 (AtaxiaSubtype)

5.1 Cerebellum

Distribution of sample RIN for Cerebellum level 2 study:

KnownAtaxia

Distances (N)

Expand details
  • X-Axis: Distance between the novel splice site and the reference splice site.
  • Y-Axis: Number of unique novel junctions. Bars are stacked.
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details
  • X-Axis: Distance between the novel splice site and the reference splice site.
  • Y-Axis: Number of unique novel junctions.
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor) and cluster (same as colour).

Expand details
  • X-Axis: Distance between the novel splice site and the reference splice site.
  • Y-Axis: Difference between the number of unique novel junctions from case samples and from control samples.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor).

Modulo 3

Expand details
  • X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
  • Y-Axis: Number of unique novel junctions. Also represented as the text inside the bars.
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details
  • X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
  • Y-Axis: Percentage of each modulo 3 value with respect to cluster (case and control) and novel type (donor and acceptor).
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details
  • X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
  • Y-Axis: Percentage of each modulo 3 value with respect to cluster (case and control), novel type (donor and acceptor) and sequence (intronic and exonic).
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor) and sequence (intronic vs. exonic).

Delta MaxEntScan

Expand details
  • X-Axis: Difference between the MaxEntScan score of the reference annotated intron and the novel junction.
  • Y-Axis: Kernel density estimate (smoothed version of a histogram).
  • Colour: Cluster type as case vs. control.
  • Facets: Different splice sites.

Expand details
  • X-Axis: Difference between the MaxEntScan score of the reference annotated intron and the novel junction.
  • Y-Axis: Difference between the kernel density estimate for case samples and control samples.
  • Colour: Cluster type as case vs. control.
  • Facets: Different splice sites.

MSR

## <br><br><br>

Expand details
  • X-Axis: The mis-splicing ratio.
  • Y-Axis: Kernel density estimate.
  • Colour: Cluster type as case vs. control.
  • Facets: Different splice sites.
  • Notes: Distributions have a discontinuity at X = 0.05 to better represent all possible MSR values. In some cases, a discontinuity in the Y-axis is also present if the difference between the two sample types exceeds a predefined threshold.

Stats

Unique annotated introns
Mis-spliced site Cluster # Annotated introns # Ann. introns by splice site Percent [%]
acceptor Control 195965 38514 19.65
acceptor KnownAtaxia 195965 36451 18.60
both Control 195965 31939 16.30
both KnownAtaxia 195965 27608 14.09
donor Control 195965 21431 10.94
donor KnownAtaxia 195965 21444 10.94
never Control 195965 104081 53.11
never KnownAtaxia 195965 110462 56.37
Expand details
  • # Annotated introns: number of total annotated introns in the cluster. Since we limit the annotated introns to those common across all samples and clusters, the number is the same for both clusters.
  • # Ann. introns by splice site: the number of annotated introns for the cluster and splice site.
  • Percent [%]: the percentage that the annotated introns in the splice site represent to the total number of introns in the cluster:
\[ \text{Percent [%]}=\frac{\text{# Ann. introns by splice site}}{\text{# Annotated introns}}*100\% \]

Reads - annotated introns
Cluster # Annotated introns # Reads in cluster # Ann. intron reads Percentage [%]
Case 195965 3448881421 324372917 9.41
Control 195965 3977174051 432584614 10.88
Expand details
  • # Annotated introns: number of common annotated introns in the cluster. Since we limit the annotated introns to those common across all samples and clusters, the number is the same for both clusters.
  • # Reads in cluster: total number of reads from the BAM files that constitutes the cluster.
  • # Ann. intron reads: reads associated to the annotated introns in the cluster. Only common introns are being counted.
  • Percent [%]: the percentage that the annotated intron reads represent to the total number of reads in the cluster.
\[ \text{Percent [%]}=\frac{\text{# Ann. intron reads}}{\text{# Reads in cluster}}*100\% \]

Reads - novel junctions
Cluster Novel type # Novel junctions # Novel junc. in splice site # Reads in cluster # Novel junc. reads Percentage [%]
Case novel_acceptor 189450 108616 3448881421 1741540 0.05
Case novel_donor 189450 80834 3448881421 1260992 0.04
Control novel_acceptor 221459 128194 3977174051 2379406 0.06
Control novel_donor 221459 93265 3977174051 1688372 0.04
Expand details
  • # Novel junctions: total number of novel junctions for that particular cluster.
  • # Novel junc. in splice site: total number of novel junctions for that particular splice site in the cluster.
  • # Reads in cluster: total number of reads from the BAM files that constitutes the cluster.
  • # Novel junc. reads: reads associated to the novel junctions in the cluster and splice site.
  • Percent [%]: the percentage that the novel junction reads represent to the total number of reads in the cluster.
\[ \text{Percent [%]}=\frac{\text{# Novel junc. reads}}{\text{# Reads in cluster}}*100\% \]

UnknownAtaxia

Distances (N)

Expand details
  • X-Axis: Distance between the novel splice site and the reference splice site.
  • Y-Axis: Number of unique novel junctions. Bars are stacked.
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details
  • X-Axis: Distance between the novel splice site and the reference splice site.
  • Y-Axis: Number of unique novel junctions.
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor) and cluster (same as colour).

Expand details
  • X-Axis: Distance between the novel splice site and the reference splice site.
  • Y-Axis: Difference between the number of unique novel junctions from case samples and from control samples.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor).

Modulo 3

Expand details
  • X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
  • Y-Axis: Number of unique novel junctions. Also represented as the text inside the bars.
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details
  • X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
  • Y-Axis: Percentage of each modulo 3 value with respect to cluster (case and control) and novel type (donor and acceptor).
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details
  • X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
  • Y-Axis: Percentage of each modulo 3 value with respect to cluster (case and control), novel type (donor and acceptor) and sequence (intronic and exonic).
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor) and sequence (intronic vs. exonic).

Delta MaxEntScan

Expand details
  • X-Axis: Difference between the MaxEntScan score of the reference annotated intron and the novel junction.
  • Y-Axis: Kernel density estimate (smoothed version of a histogram).
  • Colour: Cluster type as case vs. control.
  • Facets: Different splice sites.

Expand details
  • X-Axis: Difference between the MaxEntScan score of the reference annotated intron and the novel junction.
  • Y-Axis: Difference between the kernel density estimate for case samples and control samples.
  • Colour: Cluster type as case vs. control.
  • Facets: Different splice sites.

MSR

## <br><br><br>

Expand details
  • X-Axis: The mis-splicing ratio.
  • Y-Axis: Kernel density estimate.
  • Colour: Cluster type as case vs. control.
  • Facets: Different splice sites.
  • Notes: Distributions have a discontinuity at X = 0.05 to better represent all possible MSR values. In some cases, a discontinuity in the Y-axis is also present if the difference between the two sample types exceeds a predefined threshold.

Stats

Unique annotated introns
Mis-spliced site Cluster # Annotated introns # Ann. introns by splice site Percent [%]
acceptor Control 182348 26187 14.36
acceptor UnknownAtaxia 182348 31827 17.45
both Control 182348 14442 7.92
both UnknownAtaxia 182348 19905 10.92
donor Control 182348 16653 9.13
donor UnknownAtaxia 182348 17614 9.66
never Control 182348 125066 68.59
never UnknownAtaxia 182348 113002 61.97
Expand details
  • # Annotated introns: number of total annotated introns in the cluster. Since we limit the annotated introns to those common across all samples and clusters, the number is the same for both clusters.
  • # Ann. introns by splice site: the number of annotated introns for the cluster and splice site.
  • Percent [%]: the percentage that the annotated introns in the splice site represent to the total number of introns in the cluster:
\[ \text{Percent [%]}=\frac{\text{# Ann. introns by splice site}}{\text{# Annotated introns}}*100\% \]

Reads - annotated introns
Cluster # Annotated introns # Reads in cluster # Ann. intron reads Percentage [%]
Case 182348 1723566342 159025954 9.23
Control 182348 1693165552 136668669 8.07
Expand details
  • # Annotated introns: number of common annotated introns in the cluster. Since we limit the annotated introns to those common across all samples and clusters, the number is the same for both clusters.
  • # Reads in cluster: total number of reads from the BAM files that constitutes the cluster.
  • # Ann. intron reads: reads associated to the annotated introns in the cluster. Only common introns are being counted.
  • Percent [%]: the percentage that the annotated intron reads represent to the total number of reads in the cluster.
\[ \text{Percent [%]}=\frac{\text{# Ann. intron reads}}{\text{# Reads in cluster}}*100\% \]

Reads - novel junctions
Cluster Novel type # Novel junctions # Novel junc. in splice site # Reads in cluster # Novel junc. reads Percentage [%]
Case novel_acceptor 137582 80978 1723566342 856784 0.05
Case novel_donor 137582 56604 1723566342 612918 0.04
Control novel_acceptor 102863 58382 1693165552 669761 0.04
Control novel_donor 102863 44481 1693165552 458738 0.03
Expand details
  • # Novel junctions: total number of novel junctions for that particular cluster.
  • # Novel junc. in splice site: total number of novel junctions for that particular splice site in the cluster.
  • # Reads in cluster: total number of reads from the BAM files that constitutes the cluster.
  • # Novel junc. reads: reads associated to the novel junctions in the cluster and splice site.
  • Percent [%]: the percentage that the novel junction reads represent to the total number of reads in the cluster.
\[ \text{Percent [%]}=\frac{\text{# Novel junc. reads}}{\text{# Reads in cluster}}*100\% \]

5.2 Frontal Cortex

Distribution of sample RIN for Frontal Cortex level 2 study:

KnownAtaxia

Distances (N)

Expand details
  • X-Axis: Distance between the novel splice site and the reference splice site.
  • Y-Axis: Number of unique novel junctions. Bars are stacked.
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details
  • X-Axis: Distance between the novel splice site and the reference splice site.
  • Y-Axis: Number of unique novel junctions.
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor) and cluster (same as colour).

Expand details
  • X-Axis: Distance between the novel splice site and the reference splice site.
  • Y-Axis: Difference between the number of unique novel junctions from case samples and from control samples.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor).

Modulo 3

Expand details
  • X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
  • Y-Axis: Number of unique novel junctions. Also represented as the text inside the bars.
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details
  • X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
  • Y-Axis: Percentage of each modulo 3 value with respect to cluster (case and control) and novel type (donor and acceptor).
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details
  • X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
  • Y-Axis: Percentage of each modulo 3 value with respect to cluster (case and control), novel type (donor and acceptor) and sequence (intronic and exonic).
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor) and sequence (intronic vs. exonic).

Delta MaxEntScan

Expand details
  • X-Axis: Difference between the MaxEntScan score of the reference annotated intron and the novel junction.
  • Y-Axis: Kernel density estimate (smoothed version of a histogram).
  • Colour: Cluster type as case vs. control.
  • Facets: Different splice sites.

Expand details
  • X-Axis: Difference between the MaxEntScan score of the reference annotated intron and the novel junction.
  • Y-Axis: Difference between the kernel density estimate for case samples and control samples.
  • Colour: Cluster type as case vs. control.
  • Facets: Different splice sites.

MSR

## <br><br><br>

Expand details
  • X-Axis: The mis-splicing ratio.
  • Y-Axis: Kernel density estimate.
  • Colour: Cluster type as case vs. control.
  • Facets: Different splice sites.
  • Notes: Distributions have a discontinuity at X = 0.05 to better represent all possible MSR values. In some cases, a discontinuity in the Y-axis is also present if the difference between the two sample types exceeds a predefined threshold.

Stats

Unique annotated introns
Mis-spliced site Cluster # Annotated introns # Ann. introns by splice site Percent [%]
acceptor Control 209708 40473 19.30
acceptor KnownAtaxia 209708 41017 19.56
both Control 209708 34187 16.30
both KnownAtaxia 209708 34357 16.38
donor Control 209708 24851 11.85
donor KnownAtaxia 209708 25050 11.95
never Control 209708 110197 52.55
never KnownAtaxia 209708 109284 52.11
Expand details
  • # Annotated introns: number of total annotated introns in the cluster. Since we limit the annotated introns to those common across all samples and clusters, the number is the same for both clusters.
  • # Ann. introns by splice site: the number of annotated introns for the cluster and splice site.
  • Percent [%]: the percentage that the annotated introns in the splice site represent to the total number of introns in the cluster:
\[ \text{Percent [%]}=\frac{\text{# Ann. introns by splice site}}{\text{# Annotated introns}}*100\% \]

Reads - annotated introns
Cluster # Annotated introns # Reads in cluster # Ann. intron reads Percentage [%]
Case 209708 3572908572 665252255 18.62
Control 209708 4100844167 702866511 17.14
Expand details
  • # Annotated introns: number of common annotated introns in the cluster. Since we limit the annotated introns to those common across all samples and clusters, the number is the same for both clusters.
  • # Reads in cluster: total number of reads from the BAM files that constitutes the cluster.
  • # Ann. intron reads: reads associated to the annotated introns in the cluster. Only common introns are being counted.
  • Percent [%]: the percentage that the annotated intron reads represent to the total number of reads in the cluster.
\[ \text{Percent [%]}=\frac{\text{# Ann. intron reads}}{\text{# Reads in cluster}}*100\% \]

Reads - novel junctions
Cluster Novel type # Novel junctions # Novel junc. in splice site # Reads in cluster # Novel junc. reads Percentage [%]
Case novel_acceptor 241427 134997 3572908572 2283057 0.06
Case novel_donor 241427 106430 3572908572 1709242 0.05
Control novel_acceptor 239218 133056 4100844167 2464214 0.06
Control novel_donor 239218 106162 4100844167 1849022 0.05
Expand details
  • # Novel junctions: total number of novel junctions for that particular cluster.
  • # Novel junc. in splice site: total number of novel junctions for that particular splice site in the cluster.
  • # Reads in cluster: total number of reads from the BAM files that constitutes the cluster.
  • # Novel junc. reads: reads associated to the novel junctions in the cluster and splice site.
  • Percent [%]: the percentage that the novel junction reads represent to the total number of reads in the cluster.
\[ \text{Percent [%]}=\frac{\text{# Novel junc. reads}}{\text{# Reads in cluster}}*100\% \]

UnknownAtaxia

Distances (N)

Expand details
  • X-Axis: Distance between the novel splice site and the reference splice site.
  • Y-Axis: Number of unique novel junctions. Bars are stacked.
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details
  • X-Axis: Distance between the novel splice site and the reference splice site.
  • Y-Axis: Number of unique novel junctions.
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor) and cluster (same as colour).

Expand details
  • X-Axis: Distance between the novel splice site and the reference splice site.
  • Y-Axis: Difference between the number of unique novel junctions from case samples and from control samples.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor).

Modulo 3

Expand details
  • X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
  • Y-Axis: Number of unique novel junctions. Also represented as the text inside the bars.
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details
  • X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
  • Y-Axis: Percentage of each modulo 3 value with respect to cluster (case and control) and novel type (donor and acceptor).
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details
  • X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
  • Y-Axis: Percentage of each modulo 3 value with respect to cluster (case and control), novel type (donor and acceptor) and sequence (intronic and exonic).
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor) and sequence (intronic vs. exonic).

Delta MaxEntScan

Expand details
  • X-Axis: Difference between the MaxEntScan score of the reference annotated intron and the novel junction.
  • Y-Axis: Kernel density estimate (smoothed version of a histogram).
  • Colour: Cluster type as case vs. control.
  • Facets: Different splice sites.

Expand details
  • X-Axis: Difference between the MaxEntScan score of the reference annotated intron and the novel junction.
  • Y-Axis: Difference between the kernel density estimate for case samples and control samples.
  • Colour: Cluster type as case vs. control.
  • Facets: Different splice sites.

MSR

## <br><br><br>

Expand details
  • X-Axis: The mis-splicing ratio.
  • Y-Axis: Kernel density estimate.
  • Colour: Cluster type as case vs. control.
  • Facets: Different splice sites.
  • Notes: Distributions have a discontinuity at X = 0.05 to better represent all possible MSR values. In some cases, a discontinuity in the Y-axis is also present if the difference between the two sample types exceeds a predefined threshold.

Stats

Unique annotated introns
Mis-spliced site Cluster # Annotated introns # Ann. introns by splice site Percent [%]
acceptor Control 195328 31309 16.03
acceptor UnknownAtaxia 195328 30651 15.69
both Control 195328 19848 10.16
both UnknownAtaxia 195328 17533 8.98
donor Control 195328 20335 10.41
donor UnknownAtaxia 195328 19693 10.08
never Control 195328 123836 63.40
never UnknownAtaxia 195328 127451 65.25
Expand details
  • # Annotated introns: number of total annotated introns in the cluster. Since we limit the annotated introns to those common across all samples and clusters, the number is the same for both clusters.
  • # Ann. introns by splice site: the number of annotated introns for the cluster and splice site.
  • Percent [%]: the percentage that the annotated introns in the splice site represent to the total number of introns in the cluster:
\[ \text{Percent [%]}=\frac{\text{# Ann. introns by splice site}}{\text{# Annotated introns}}*100\% \]

Reads - annotated introns
Cluster # Annotated introns # Reads in cluster # Ann. intron reads Percentage [%]
Case 195328 1659144178 253102013 15.25
Control 195328 1765693058 293450631 16.62
Expand details
  • # Annotated introns: number of common annotated introns in the cluster. Since we limit the annotated introns to those common across all samples and clusters, the number is the same for both clusters.
  • # Reads in cluster: total number of reads from the BAM files that constitutes the cluster.
  • # Ann. intron reads: reads associated to the annotated introns in the cluster. Only common introns are being counted.
  • Percent [%]: the percentage that the annotated intron reads represent to the total number of reads in the cluster.
\[ \text{Percent [%]}=\frac{\text{# Ann. intron reads}}{\text{# Reads in cluster}}*100\% \]

Reads - novel junctions
Cluster Novel type # Novel junctions # Novel junc. in splice site # Reads in cluster # Novel junc. reads Percentage [%]
Case novel_acceptor 126819 71270 1659144178 833338 0.05
Case novel_donor 126819 55549 1659144178 618577 0.04
Control novel_acceptor 140907 78017 1765693058 972618 0.06
Control novel_donor 140907 62890 1765693058 760132 0.04
Expand details
  • # Novel junctions: total number of novel junctions for that particular cluster.
  • # Novel junc. in splice site: total number of novel junctions for that particular splice site in the cluster.
  • # Reads in cluster: total number of reads from the BAM files that constitutes the cluster.
  • # Novel junc. reads: reads associated to the novel junctions in the cluster and splice site.
  • Percent [%]: the percentage that the novel junction reads represent to the total number of reads in the cluster.
\[ \text{Percent [%]}=\frac{\text{# Novel junc. reads}}{\text{# Reads in cluster}}*100\% \]

6 Results for Level 3 (Diagnosis)

6.1 Cerebellum

Distribution of sample RIN for Cerebellum level 3 study:

FRDA

Distances (N)

Expand details
  • X-Axis: Distance between the novel splice site and the reference splice site.
  • Y-Axis: Number of unique novel junctions. Bars are stacked.
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details
  • X-Axis: Distance between the novel splice site and the reference splice site.
  • Y-Axis: Number of unique novel junctions.
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor) and cluster (same as colour).

Expand details
  • X-Axis: Distance between the novel splice site and the reference splice site.
  • Y-Axis: Difference between the number of unique novel junctions from case samples and from control samples.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor).

Modulo 3

Expand details
  • X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
  • Y-Axis: Number of unique novel junctions. Also represented as the text inside the bars.
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details
  • X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
  • Y-Axis: Percentage of each modulo 3 value with respect to cluster (case and control) and novel type (donor and acceptor).
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details
  • X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
  • Y-Axis: Percentage of each modulo 3 value with respect to cluster (case and control), novel type (donor and acceptor) and sequence (intronic and exonic).
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor) and sequence (intronic vs. exonic).

Delta MaxEntScan

Expand details
  • X-Axis: Difference between the MaxEntScan score of the reference annotated intron and the novel junction.
  • Y-Axis: Kernel density estimate (smoothed version of a histogram).
  • Colour: Cluster type as case vs. control.
  • Facets: Different splice sites.

Expand details
  • X-Axis: Difference between the MaxEntScan score of the reference annotated intron and the novel junction.
  • Y-Axis: Difference between the kernel density estimate for case samples and control samples.
  • Colour: Cluster type as case vs. control.
  • Facets: Different splice sites.

MSR

## <br><br><br>

Expand details
  • X-Axis: The mis-splicing ratio.
  • Y-Axis: Kernel density estimate.
  • Colour: Cluster type as case vs. control.
  • Facets: Different splice sites.
  • Notes: Distributions have a discontinuity at X = 0.05 to better represent all possible MSR values. In some cases, a discontinuity in the Y-axis is also present if the difference between the two sample types exceeds a predefined threshold.

Stats

Unique annotated introns
Mis-spliced site Cluster # Annotated introns # Ann. introns by splice site Percent [%]
acceptor Control 176181 23705 13.45
acceptor FRDA 176181 24542 13.93
both Control 176181 12569 7.13
both FRDA 176181 13221 7.50
donor Control 176181 15157 8.60
donor FRDA 176181 15665 8.89
never Control 176181 124750 70.81
never FRDA 176181 122753 69.67
Expand details
  • # Annotated introns: number of total annotated introns in the cluster. Since we limit the annotated introns to those common across all samples and clusters, the number is the same for both clusters.
  • # Ann. introns by splice site: the number of annotated introns for the cluster and splice site.
  • Percent [%]: the percentage that the annotated introns in the splice site represent to the total number of introns in the cluster:
\[ \text{Percent [%]}=\frac{\text{# Ann. introns by splice site}}{\text{# Annotated introns}}*100\% \]

Reads - annotated introns
Cluster # Annotated introns # Reads in cluster # Ann. intron reads Percentage [%]
Case 176181 896665067 89994863 10.04
Control 176181 937441018 94447687 10.08
Expand details
  • # Annotated introns: number of common annotated introns in the cluster. Since we limit the annotated introns to those common across all samples and clusters, the number is the same for both clusters.
  • # Reads in cluster: total number of reads from the BAM files that constitutes the cluster.
  • # Ann. intron reads: reads associated to the annotated introns in the cluster. Only common introns are being counted.
  • Percent [%]: the percentage that the annotated intron reads represent to the total number of reads in the cluster.
\[ \text{Percent [%]}=\frac{\text{# Ann. intron reads}}{\text{# Reads in cluster}}*100\% \]

Reads - novel junctions
Cluster Novel type # Novel junctions # Novel junc. in splice site # Reads in cluster # Novel junc. reads Percentage [%]
Case novel_acceptor 93579 53271 896665067 527632 0.06
Case novel_donor 93579 40308 896665067 377082 0.04
Control novel_acceptor 89193 50708 937441018 508712 0.05
Control novel_donor 89193 38485 937441018 349410 0.04
Expand details
  • # Novel junctions: total number of novel junctions for that particular cluster.
  • # Novel junc. in splice site: total number of novel junctions for that particular splice site in the cluster.
  • # Reads in cluster: total number of reads from the BAM files that constitutes the cluster.
  • # Novel junc. reads: reads associated to the novel junctions in the cluster and splice site.
  • Percent [%]: the percentage that the novel junction reads represent to the total number of reads in the cluster.
\[ \text{Percent [%]}=\frac{\text{# Novel junc. reads}}{\text{# Reads in cluster}}*100\% \]

SCA1

Distances (N)

Expand details
  • X-Axis: Distance between the novel splice site and the reference splice site.
  • Y-Axis: Number of unique novel junctions. Bars are stacked.
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details
  • X-Axis: Distance between the novel splice site and the reference splice site.
  • Y-Axis: Number of unique novel junctions.
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor) and cluster (same as colour).

Expand details
  • X-Axis: Distance between the novel splice site and the reference splice site.
  • Y-Axis: Difference between the number of unique novel junctions from case samples and from control samples.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor).

Modulo 3

Expand details
  • X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
  • Y-Axis: Number of unique novel junctions. Also represented as the text inside the bars.
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details
  • X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
  • Y-Axis: Percentage of each modulo 3 value with respect to cluster (case and control) and novel type (donor and acceptor).
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details
  • X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
  • Y-Axis: Percentage of each modulo 3 value with respect to cluster (case and control), novel type (donor and acceptor) and sequence (intronic and exonic).
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor) and sequence (intronic vs. exonic).

Delta MaxEntScan

Expand details
  • X-Axis: Difference between the MaxEntScan score of the reference annotated intron and the novel junction.
  • Y-Axis: Kernel density estimate (smoothed version of a histogram).
  • Colour: Cluster type as case vs. control.
  • Facets: Different splice sites.

Expand details
  • X-Axis: Difference between the MaxEntScan score of the reference annotated intron and the novel junction.
  • Y-Axis: Difference between the kernel density estimate for case samples and control samples.
  • Colour: Cluster type as case vs. control.
  • Facets: Different splice sites.

MSR

## <br><br><br>

Expand details
  • X-Axis: The mis-splicing ratio.
  • Y-Axis: Kernel density estimate.
  • Colour: Cluster type as case vs. control.
  • Facets: Different splice sites.
  • Notes: Distributions have a discontinuity at X = 0.05 to better represent all possible MSR values. In some cases, a discontinuity in the Y-axis is also present if the difference between the two sample types exceeds a predefined threshold.

Stats

Unique annotated introns
Mis-spliced site Cluster # Annotated introns # Ann. introns by splice site Percent [%]
acceptor Control 168115 19582 11.65
acceptor SCA1 168115 18882 11.23
both Control 168115 9200 5.47
both SCA1 168115 8590 5.11
donor Control 168115 12736 7.58
donor SCA1 168115 13153 7.82
never Control 168115 126597 75.30
never SCA1 168115 127490 75.83
Expand details
  • # Annotated introns: number of total annotated introns in the cluster. Since we limit the annotated introns to those common across all samples and clusters, the number is the same for both clusters.
  • # Ann. introns by splice site: the number of annotated introns for the cluster and splice site.
  • Percent [%]: the percentage that the annotated introns in the splice site represent to the total number of introns in the cluster:
\[ \text{Percent [%]}=\frac{\text{# Ann. introns by splice site}}{\text{# Annotated introns}}*100\% \]

Reads - annotated introns
Cluster # Annotated introns # Reads in cluster # Ann. intron reads Percentage [%]
Case 168115 663185400 55447132 8.36
Control 168115 727022405 60692971 8.35
Expand details
  • # Annotated introns: number of common annotated introns in the cluster. Since we limit the annotated introns to those common across all samples and clusters, the number is the same for both clusters.
  • # Reads in cluster: total number of reads from the BAM files that constitutes the cluster.
  • # Ann. intron reads: reads associated to the annotated introns in the cluster. Only common introns are being counted.
  • Percent [%]: the percentage that the annotated intron reads represent to the total number of reads in the cluster.
\[ \text{Percent [%]}=\frac{\text{# Ann. intron reads}}{\text{# Reads in cluster}}*100\% \]

Reads - novel junctions
Cluster Novel type # Novel junctions # Novel junc. in splice site # Reads in cluster # Novel junc. reads Percentage [%]
Case novel_acceptor 64141 35767 663185400 281691 0.04
Case novel_donor 64141 28374 663185400 210850 0.03
Control novel_acceptor 67003 38156 727022405 327377 0.05
Control novel_donor 67003 28847 727022405 233144 0.03
Expand details
  • # Novel junctions: total number of novel junctions for that particular cluster.
  • # Novel junc. in splice site: total number of novel junctions for that particular splice site in the cluster.
  • # Reads in cluster: total number of reads from the BAM files that constitutes the cluster.
  • # Novel junc. reads: reads associated to the novel junctions in the cluster and splice site.
  • Percent [%]: the percentage that the novel junction reads represent to the total number of reads in the cluster.
\[ \text{Percent [%]}=\frac{\text{# Novel junc. reads}}{\text{# Reads in cluster}}*100\% \]

SCA2

Distances (N)

Expand details
  • X-Axis: Distance between the novel splice site and the reference splice site.
  • Y-Axis: Number of unique novel junctions. Bars are stacked.
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details
  • X-Axis: Distance between the novel splice site and the reference splice site.
  • Y-Axis: Number of unique novel junctions.
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor) and cluster (same as colour).

Expand details
  • X-Axis: Distance between the novel splice site and the reference splice site.
  • Y-Axis: Difference between the number of unique novel junctions from case samples and from control samples.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor).

Modulo 3

Expand details
  • X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
  • Y-Axis: Number of unique novel junctions. Also represented as the text inside the bars.
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details
  • X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
  • Y-Axis: Percentage of each modulo 3 value with respect to cluster (case and control) and novel type (donor and acceptor).
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details
  • X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
  • Y-Axis: Percentage of each modulo 3 value with respect to cluster (case and control), novel type (donor and acceptor) and sequence (intronic and exonic).
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor) and sequence (intronic vs. exonic).

Delta MaxEntScan

Expand details
  • X-Axis: Difference between the MaxEntScan score of the reference annotated intron and the novel junction.
  • Y-Axis: Kernel density estimate (smoothed version of a histogram).
  • Colour: Cluster type as case vs. control.
  • Facets: Different splice sites.

Expand details
  • X-Axis: Difference between the MaxEntScan score of the reference annotated intron and the novel junction.
  • Y-Axis: Difference between the kernel density estimate for case samples and control samples.
  • Colour: Cluster type as case vs. control.
  • Facets: Different splice sites.

MSR

## <br><br><br>

Expand details
  • X-Axis: The mis-splicing ratio.
  • Y-Axis: Kernel density estimate.
  • Colour: Cluster type as case vs. control.
  • Facets: Different splice sites.
  • Notes: Distributions have a discontinuity at X = 0.05 to better represent all possible MSR values. In some cases, a discontinuity in the Y-axis is also present if the difference between the two sample types exceeds a predefined threshold.

Stats

Unique annotated introns
Mis-spliced site Cluster # Annotated introns # Ann. introns by splice site Percent [%]
acceptor Control 159886 13505 8.45
acceptor SCA2 159886 14135 8.84
both Control 159886 4523 2.83
both SCA2 159886 4381 2.74
donor Control 159886 9123 5.71
donor SCA2 159886 9079 5.68
never Control 159886 132735 83.02
never SCA2 159886 132291 82.74
Expand details
  • # Annotated introns: number of total annotated introns in the cluster. Since we limit the annotated introns to those common across all samples and clusters, the number is the same for both clusters.
  • # Ann. introns by splice site: the number of annotated introns for the cluster and splice site.
  • Percent [%]: the percentage that the annotated introns in the splice site represent to the total number of introns in the cluster:
\[ \text{Percent [%]}=\frac{\text{# Ann. introns by splice site}}{\text{# Annotated introns}}*100\% \]

Reads - annotated introns
Cluster # Annotated introns # Reads in cluster # Ann. intron reads Percentage [%]
Case 159886 584147295 38935749 6.67
Control 159886 710683525 39604626 5.57
Expand details
  • # Annotated introns: number of common annotated introns in the cluster. Since we limit the annotated introns to those common across all samples and clusters, the number is the same for both clusters.
  • # Reads in cluster: total number of reads from the BAM files that constitutes the cluster.
  • # Ann. intron reads: reads associated to the annotated introns in the cluster. Only common introns are being counted.
  • Percent [%]: the percentage that the annotated intron reads represent to the total number of reads in the cluster.
\[ \text{Percent [%]}=\frac{\text{# Ann. intron reads}}{\text{# Reads in cluster}}*100\% \]

Reads - novel junctions
Cluster Novel type # Novel junctions # Novel junc. in splice site # Reads in cluster # Novel junc. reads Percentage [%]
Case novel_acceptor 39416 22961 584147295 182180 0.03
Case novel_donor 39416 16455 584147295 132288 0.02
Control novel_acceptor 38531 21928 710683525 169624 0.02
Control novel_donor 38531 16603 710683525 118165 0.02
Expand details
  • # Novel junctions: total number of novel junctions for that particular cluster.
  • # Novel junc. in splice site: total number of novel junctions for that particular splice site in the cluster.
  • # Reads in cluster: total number of reads from the BAM files that constitutes the cluster.
  • # Novel junc. reads: reads associated to the novel junctions in the cluster and splice site.
  • Percent [%]: the percentage that the novel junction reads represent to the total number of reads in the cluster.
\[ \text{Percent [%]}=\frac{\text{# Novel junc. reads}}{\text{# Reads in cluster}}*100\% \]

SCA6

Distances (N)

Expand details
  • X-Axis: Distance between the novel splice site and the reference splice site.
  • Y-Axis: Number of unique novel junctions. Bars are stacked.
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details
  • X-Axis: Distance between the novel splice site and the reference splice site.
  • Y-Axis: Number of unique novel junctions.
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor) and cluster (same as colour).

Expand details
  • X-Axis: Distance between the novel splice site and the reference splice site.
  • Y-Axis: Difference between the number of unique novel junctions from case samples and from control samples.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor).

Modulo 3

Expand details
  • X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
  • Y-Axis: Number of unique novel junctions. Also represented as the text inside the bars.
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details
  • X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
  • Y-Axis: Percentage of each modulo 3 value with respect to cluster (case and control) and novel type (donor and acceptor).
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details
  • X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
  • Y-Axis: Percentage of each modulo 3 value with respect to cluster (case and control), novel type (donor and acceptor) and sequence (intronic and exonic).
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor) and sequence (intronic vs. exonic).

Delta MaxEntScan

Expand details
  • X-Axis: Difference between the MaxEntScan score of the reference annotated intron and the novel junction.
  • Y-Axis: Kernel density estimate (smoothed version of a histogram).
  • Colour: Cluster type as case vs. control.
  • Facets: Different splice sites.

Expand details
  • X-Axis: Difference between the MaxEntScan score of the reference annotated intron and the novel junction.
  • Y-Axis: Difference between the kernel density estimate for case samples and control samples.
  • Colour: Cluster type as case vs. control.
  • Facets: Different splice sites.

MSR

## <br><br><br>

Expand details
  • X-Axis: The mis-splicing ratio.
  • Y-Axis: Kernel density estimate.
  • Colour: Cluster type as case vs. control.
  • Facets: Different splice sites.
  • Notes: Distributions have a discontinuity at X = 0.05 to better represent all possible MSR values. In some cases, a discontinuity in the Y-axis is also present if the difference between the two sample types exceeds a predefined threshold.

Stats

Unique annotated introns
Mis-spliced site Cluster # Annotated introns # Ann. introns by splice site Percent [%]
acceptor Control 170971 20916 12.23
acceptor SCA6 170971 19858 11.61
both Control 170971 10418 6.09
both SCA6 170971 8296 4.85
donor Control 170971 13521 7.91
donor SCA6 170971 12953 7.58
never Control 170971 126116 73.76
never SCA6 170971 129864 75.96
Expand details
  • # Annotated introns: number of total annotated introns in the cluster. Since we limit the annotated introns to those common across all samples and clusters, the number is the same for both clusters.
  • # Ann. introns by splice site: the number of annotated introns for the cluster and splice site.
  • Percent [%]: the percentage that the annotated introns in the splice site represent to the total number of introns in the cluster:
\[ \text{Percent [%]}=\frac{\text{# Ann. introns by splice site}}{\text{# Annotated introns}}*100\% \]

Reads - annotated introns
Cluster # Annotated introns # Reads in cluster # Ann. intron reads Percentage [%]
Case 170971 616156554 67782298 11.00
Control 170971 712278697 68984361 9.69
Expand details
  • # Annotated introns: number of common annotated introns in the cluster. Since we limit the annotated introns to those common across all samples and clusters, the number is the same for both clusters.
  • # Reads in cluster: total number of reads from the BAM files that constitutes the cluster.
  • # Ann. intron reads: reads associated to the annotated introns in the cluster. Only common introns are being counted.
  • Percent [%]: the percentage that the annotated intron reads represent to the total number of reads in the cluster.
\[ \text{Percent [%]}=\frac{\text{# Ann. intron reads}}{\text{# Reads in cluster}}*100\% \]

Reads - novel junctions
Cluster Novel type # Novel junctions # Novel junc. in splice site # Reads in cluster # Novel junc. reads Percentage [%]
Case novel_acceptor 64023 36427 616156554 316179 0.05
Case novel_donor 64023 27596 616156554 220631 0.04
Control novel_acceptor 74679 42471 712278697 403422 0.06
Control novel_donor 74679 32208 712278697 275237 0.04
Expand details
  • # Novel junctions: total number of novel junctions for that particular cluster.
  • # Novel junc. in splice site: total number of novel junctions for that particular splice site in the cluster.
  • # Reads in cluster: total number of reads from the BAM files that constitutes the cluster.
  • # Novel junc. reads: reads associated to the novel junctions in the cluster and splice site.
  • Percent [%]: the percentage that the novel junction reads represent to the total number of reads in the cluster.
\[ \text{Percent [%]}=\frac{\text{# Novel junc. reads}}{\text{# Reads in cluster}}*100\% \]

6.2 Frontal Cortex

Distribution of sample RIN for Frontal Cortex level 3 study:

FRDA

Distances (N)

Expand details
  • X-Axis: Distance between the novel splice site and the reference splice site.
  • Y-Axis: Number of unique novel junctions. Bars are stacked.
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details
  • X-Axis: Distance between the novel splice site and the reference splice site.
  • Y-Axis: Number of unique novel junctions.
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor) and cluster (same as colour).

Expand details
  • X-Axis: Distance between the novel splice site and the reference splice site.
  • Y-Axis: Difference between the number of unique novel junctions from case samples and from control samples.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor).

Modulo 3

Expand details
  • X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
  • Y-Axis: Number of unique novel junctions. Also represented as the text inside the bars.
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details
  • X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
  • Y-Axis: Percentage of each modulo 3 value with respect to cluster (case and control) and novel type (donor and acceptor).
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details
  • X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
  • Y-Axis: Percentage of each modulo 3 value with respect to cluster (case and control), novel type (donor and acceptor) and sequence (intronic and exonic).
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor) and sequence (intronic vs. exonic).

Delta MaxEntScan

Expand details
  • X-Axis: Difference between the MaxEntScan score of the reference annotated intron and the novel junction.
  • Y-Axis: Kernel density estimate (smoothed version of a histogram).
  • Colour: Cluster type as case vs. control.
  • Facets: Different splice sites.

Expand details
  • X-Axis: Difference between the MaxEntScan score of the reference annotated intron and the novel junction.
  • Y-Axis: Difference between the kernel density estimate for case samples and control samples.
  • Colour: Cluster type as case vs. control.
  • Facets: Different splice sites.

MSR

## <br><br><br>

Expand details
  • X-Axis: The mis-splicing ratio.
  • Y-Axis: Kernel density estimate.
  • Colour: Cluster type as case vs. control.
  • Facets: Different splice sites.
  • Notes: Distributions have a discontinuity at X = 0.05 to better represent all possible MSR values. In some cases, a discontinuity in the Y-axis is also present if the difference between the two sample types exceeds a predefined threshold.

Stats

Unique annotated introns
Mis-spliced site Cluster # Annotated introns # Ann. introns by splice site Percent [%]
acceptor Control 190016 28043 14.76
acceptor FRDA 190016 26193 13.78
both Control 190016 16142 8.50
both FRDA 190016 13383 7.04
donor Control 190016 18596 9.79
donor FRDA 190016 17677 9.30
never Control 190016 127235 66.96
never FRDA 190016 132763 69.87
Expand details
  • # Annotated introns: number of total annotated introns in the cluster. Since we limit the annotated introns to those common across all samples and clusters, the number is the same for both clusters.
  • # Ann. introns by splice site: the number of annotated introns for the cluster and splice site.
  • Percent [%]: the percentage that the annotated introns in the splice site represent to the total number of introns in the cluster:
\[ \text{Percent [%]}=\frac{\text{# Ann. introns by splice site}}{\text{# Annotated introns}}*100\% \]

Reads - annotated introns
Cluster # Annotated introns # Reads in cluster # Ann. intron reads Percentage [%]
Case 190016 866370018 155141061 17.91
Control 190016 1043087110 193646726 18.56
Expand details
  • # Annotated introns: number of common annotated introns in the cluster. Since we limit the annotated introns to those common across all samples and clusters, the number is the same for both clusters.
  • # Reads in cluster: total number of reads from the BAM files that constitutes the cluster.
  • # Ann. intron reads: reads associated to the annotated introns in the cluster. Only common introns are being counted.
  • Percent [%]: the percentage that the annotated intron reads represent to the total number of reads in the cluster.
\[ \text{Percent [%]}=\frac{\text{# Ann. intron reads}}{\text{# Reads in cluster}}*100\% \]

Reads - novel junctions
Cluster Novel type # Novel junctions # Novel junc. in splice site # Reads in cluster # Novel junc. reads Percentage [%]
Case novel_acceptor 100061 55394 866370018 538635 0.06
Case novel_donor 100061 44667 866370018 402906 0.05
Control novel_acceptor 115374 63869 1043087110 696453 0.07
Control novel_donor 115374 51505 1043087110 503840 0.05
Expand details
  • # Novel junctions: total number of novel junctions for that particular cluster.
  • # Novel junc. in splice site: total number of novel junctions for that particular splice site in the cluster.
  • # Reads in cluster: total number of reads from the BAM files that constitutes the cluster.
  • # Novel junc. reads: reads associated to the novel junctions in the cluster and splice site.
  • Percent [%]: the percentage that the novel junction reads represent to the total number of reads in the cluster.
\[ \text{Percent [%]}=\frac{\text{# Novel junc. reads}}{\text{# Reads in cluster}}*100\% \]

SCA1

Distances (N)

Expand details
  • X-Axis: Distance between the novel splice site and the reference splice site.
  • Y-Axis: Number of unique novel junctions. Bars are stacked.
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details
  • X-Axis: Distance between the novel splice site and the reference splice site.
  • Y-Axis: Number of unique novel junctions.
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor) and cluster (same as colour).

Expand details
  • X-Axis: Distance between the novel splice site and the reference splice site.
  • Y-Axis: Difference between the number of unique novel junctions from case samples and from control samples.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor).

Modulo 3

Expand details
  • X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
  • Y-Axis: Number of unique novel junctions. Also represented as the text inside the bars.
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details
  • X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
  • Y-Axis: Percentage of each modulo 3 value with respect to cluster (case and control) and novel type (donor and acceptor).
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details
  • X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
  • Y-Axis: Percentage of each modulo 3 value with respect to cluster (case and control), novel type (donor and acceptor) and sequence (intronic and exonic).
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor) and sequence (intronic vs. exonic).

Delta MaxEntScan

Expand details
  • X-Axis: Difference between the MaxEntScan score of the reference annotated intron and the novel junction.
  • Y-Axis: Kernel density estimate (smoothed version of a histogram).
  • Colour: Cluster type as case vs. control.
  • Facets: Different splice sites.

Expand details
  • X-Axis: Difference between the MaxEntScan score of the reference annotated intron and the novel junction.
  • Y-Axis: Difference between the kernel density estimate for case samples and control samples.
  • Colour: Cluster type as case vs. control.
  • Facets: Different splice sites.

MSR

## <br><br><br>

Expand details
  • X-Axis: The mis-splicing ratio.
  • Y-Axis: Kernel density estimate.
  • Colour: Cluster type as case vs. control.
  • Facets: Different splice sites.
  • Notes: Distributions have a discontinuity at X = 0.05 to better represent all possible MSR values. In some cases, a discontinuity in the Y-axis is also present if the difference between the two sample types exceeds a predefined threshold.

Stats

Unique annotated introns
Mis-spliced site Cluster # Annotated introns # Ann. introns by splice site Percent [%]
acceptor Control 185853 25038 13.47
acceptor SCA1 185853 23801 12.81
both Control 185853 12990 6.99
both SCA1 185853 11645 6.27
donor Control 185853 17029 9.16
donor SCA1 185853 16604 8.93
never Control 185853 130796 70.38
never SCA1 185853 133803 71.99
Expand details
  • # Annotated introns: number of total annotated introns in the cluster. Since we limit the annotated introns to those common across all samples and clusters, the number is the same for both clusters.
  • # Ann. introns by splice site: the number of annotated introns for the cluster and splice site.
  • Percent [%]: the percentage that the annotated introns in the splice site represent to the total number of introns in the cluster:
\[ \text{Percent [%]}=\frac{\text{# Ann. introns by splice site}}{\text{# Annotated introns}}*100\% \]

Reads - annotated introns
Cluster # Annotated introns # Reads in cluster # Ann. intron reads Percentage [%]
Case 185853 698071292 143266471 20.52
Control 185853 785114845 148634904 18.93
Expand details
  • # Annotated introns: number of common annotated introns in the cluster. Since we limit the annotated introns to those common across all samples and clusters, the number is the same for both clusters.
  • # Reads in cluster: total number of reads from the BAM files that constitutes the cluster.
  • # Ann. intron reads: reads associated to the annotated introns in the cluster. Only common introns are being counted.
  • Percent [%]: the percentage that the annotated intron reads represent to the total number of reads in the cluster.
\[ \text{Percent [%]}=\frac{\text{# Ann. intron reads}}{\text{# Reads in cluster}}*100\% \]

Reads - novel junctions
Cluster Novel type # Novel junctions # Novel junc. in splice site # Reads in cluster # Novel junc. reads Percentage [%]
Case novel_acceptor 88195 48508 698071292 453261 0.06
Case novel_donor 88195 39687 698071292 345121 0.05
Control novel_acceptor 95396 52532 785114845 486387 0.06
Control novel_donor 95396 42864 785114845 370534 0.05
Expand details
  • # Novel junctions: total number of novel junctions for that particular cluster.
  • # Novel junc. in splice site: total number of novel junctions for that particular splice site in the cluster.
  • # Reads in cluster: total number of reads from the BAM files that constitutes the cluster.
  • # Novel junc. reads: reads associated to the novel junctions in the cluster and splice site.
  • Percent [%]: the percentage that the novel junction reads represent to the total number of reads in the cluster.
\[ \text{Percent [%]}=\frac{\text{# Novel junc. reads}}{\text{# Reads in cluster}}*100\% \]

SCA2

Distances (N)

Expand details
  • X-Axis: Distance between the novel splice site and the reference splice site.
  • Y-Axis: Number of unique novel junctions. Bars are stacked.
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details
  • X-Axis: Distance between the novel splice site and the reference splice site.
  • Y-Axis: Number of unique novel junctions.
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor) and cluster (same as colour).

Expand details
  • X-Axis: Distance between the novel splice site and the reference splice site.
  • Y-Axis: Difference between the number of unique novel junctions from case samples and from control samples.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor).

Modulo 3

Expand details
  • X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
  • Y-Axis: Number of unique novel junctions. Also represented as the text inside the bars.
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details
  • X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
  • Y-Axis: Percentage of each modulo 3 value with respect to cluster (case and control) and novel type (donor and acceptor).
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details
  • X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
  • Y-Axis: Percentage of each modulo 3 value with respect to cluster (case and control), novel type (donor and acceptor) and sequence (intronic and exonic).
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor) and sequence (intronic vs. exonic).

Delta MaxEntScan

Expand details
  • X-Axis: Difference between the MaxEntScan score of the reference annotated intron and the novel junction.
  • Y-Axis: Kernel density estimate (smoothed version of a histogram).
  • Colour: Cluster type as case vs. control.
  • Facets: Different splice sites.

Expand details
  • X-Axis: Difference between the MaxEntScan score of the reference annotated intron and the novel junction.
  • Y-Axis: Difference between the kernel density estimate for case samples and control samples.
  • Colour: Cluster type as case vs. control.
  • Facets: Different splice sites.

MSR

## <br><br><br>

Expand details
  • X-Axis: The mis-splicing ratio.
  • Y-Axis: Kernel density estimate.
  • Colour: Cluster type as case vs. control.
  • Facets: Different splice sites.
  • Notes: Distributions have a discontinuity at X = 0.05 to better represent all possible MSR values. In some cases, a discontinuity in the Y-axis is also present if the difference between the two sample types exceeds a predefined threshold.

Stats

Unique annotated introns
Mis-spliced site Cluster # Annotated introns # Ann. introns by splice site Percent [%]
acceptor Control 183050 24450 13.36
acceptor SCA2 183050 22490 12.29
both Control 183050 12391 6.77
both SCA2 183050 9805 5.36
donor Control 183050 16802 9.18
donor SCA2 183050 14962 8.17
never Control 183050 129407 70.69
never SCA2 183050 135793 74.18
Expand details
  • # Annotated introns: number of total annotated introns in the cluster. Since we limit the annotated introns to those common across all samples and clusters, the number is the same for both clusters.
  • # Ann. introns by splice site: the number of annotated introns for the cluster and splice site.
  • Percent [%]: the percentage that the annotated introns in the splice site represent to the total number of introns in the cluster:
\[ \text{Percent [%]}=\frac{\text{# Ann. introns by splice site}}{\text{# Annotated introns}}*100\% \]

Reads - annotated introns
Cluster # Annotated introns # Reads in cluster # Ann. intron reads Percentage [%]
Case 183050 645767566 117766633 18.24
Control 183050 762434189 141342187 18.54
Expand details
  • # Annotated introns: number of common annotated introns in the cluster. Since we limit the annotated introns to those common across all samples and clusters, the number is the same for both clusters.
  • # Reads in cluster: total number of reads from the BAM files that constitutes the cluster.
  • # Ann. intron reads: reads associated to the annotated introns in the cluster. Only common introns are being counted.
  • Percent [%]: the percentage that the annotated intron reads represent to the total number of reads in the cluster.
\[ \text{Percent [%]}=\frac{\text{# Ann. intron reads}}{\text{# Reads in cluster}}*100\% \]

Reads - novel junctions
Cluster Novel type # Novel junctions # Novel junc. in splice site # Reads in cluster # Novel junc. reads Percentage [%]
Case novel_acceptor 77025 43272 645767566 380720 0.06
Case novel_donor 77025 33753 645767566 283002 0.04
Control novel_acceptor 92338 50811 762434189 474444 0.06
Control novel_donor 92338 41527 762434189 368842 0.05
Expand details
  • # Novel junctions: total number of novel junctions for that particular cluster.
  • # Novel junc. in splice site: total number of novel junctions for that particular splice site in the cluster.
  • # Reads in cluster: total number of reads from the BAM files that constitutes the cluster.
  • # Novel junc. reads: reads associated to the novel junctions in the cluster and splice site.
  • Percent [%]: the percentage that the novel junction reads represent to the total number of reads in the cluster.
\[ \text{Percent [%]}=\frac{\text{# Novel junc. reads}}{\text{# Reads in cluster}}*100\% \]

SCA6

Distances (N)

Expand details
  • X-Axis: Distance between the novel splice site and the reference splice site.
  • Y-Axis: Number of unique novel junctions. Bars are stacked.
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details
  • X-Axis: Distance between the novel splice site and the reference splice site.
  • Y-Axis: Number of unique novel junctions.
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor) and cluster (same as colour).

Expand details
  • X-Axis: Distance between the novel splice site and the reference splice site.
  • Y-Axis: Difference between the number of unique novel junctions from case samples and from control samples.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor).

Modulo 3

Expand details
  • X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
  • Y-Axis: Number of unique novel junctions. Also represented as the text inside the bars.
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details
  • X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
  • Y-Axis: Percentage of each modulo 3 value with respect to cluster (case and control) and novel type (donor and acceptor).
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor).

Expand details
  • X-Axis: Distance in modulo 3 between the novel splice site and the reference splice site. Filtered by \(distance\leq 100\;bp\).
  • Y-Axis: Percentage of each modulo 3 value with respect to cluster (case and control), novel type (donor and acceptor) and sequence (intronic and exonic).
  • Colour: Cluster type as case vs. control.
  • Facets: Novel junction category (Novel donor vs. Novel acceptor) and sequence (intronic vs. exonic).

Delta MaxEntScan

Expand details
  • X-Axis: Difference between the MaxEntScan score of the reference annotated intron and the novel junction.
  • Y-Axis: Kernel density estimate (smoothed version of a histogram).
  • Colour: Cluster type as case vs. control.
  • Facets: Different splice sites.

Expand details
  • X-Axis: Difference between the MaxEntScan score of the reference annotated intron and the novel junction.
  • Y-Axis: Difference between the kernel density estimate for case samples and control samples.
  • Colour: Cluster type as case vs. control.
  • Facets: Different splice sites.

MSR

## <br><br><br>

Expand details
  • X-Axis: The mis-splicing ratio.
  • Y-Axis: Kernel density estimate.
  • Colour: Cluster type as case vs. control.
  • Facets: Different splice sites.
  • Notes: Distributions have a discontinuity at X = 0.05 to better represent all possible MSR values. In some cases, a discontinuity in the Y-axis is also present if the difference between the two sample types exceeds a predefined threshold.

Stats

Unique annotated introns
Mis-spliced site Cluster # Annotated introns # Ann. introns by splice site Percent [%]
acceptor Control 183595 23996 13.07
acceptor SCA6 183595 24755 13.48
both Control 183595 11435 6.23
both SCA6 183595 13080 7.12
donor Control 183595 16185 8.82
donor SCA6 183595 16434 8.95
never Control 183595 131979 71.89
never SCA6 183595 129326 70.44
Expand details
  • # Annotated introns: number of total annotated introns in the cluster. Since we limit the annotated introns to those common across all samples and clusters, the number is the same for both clusters.
  • # Ann. introns by splice site: the number of annotated introns for the cluster and splice site.
  • Percent [%]: the percentage that the annotated introns in the splice site represent to the total number of introns in the cluster:
\[ \text{Percent [%]}=\frac{\text{# Ann. introns by splice site}}{\text{# Annotated introns}}*100\% \]

Reads - annotated introns
Cluster # Annotated introns # Reads in cluster # Ann. intron reads Percentage [%]
Case 183595 669517733 120694740 18.03
Control 183595 782827004 126120311 16.11
Expand details
  • # Annotated introns: number of common annotated introns in the cluster. Since we limit the annotated introns to those common across all samples and clusters, the number is the same for both clusters.
  • # Reads in cluster: total number of reads from the BAM files that constitutes the cluster.
  • # Ann. intron reads: reads associated to the annotated introns in the cluster. Only common introns are being counted.
  • Percent [%]: the percentage that the annotated intron reads represent to the total number of reads in the cluster.
\[ \text{Percent [%]}=\frac{\text{# Ann. intron reads}}{\text{# Reads in cluster}}*100\% \]

Reads - novel junctions
Cluster Novel type # Novel junctions # Novel junc. in splice site # Reads in cluster # Novel junc. reads Percentage [%]
Case novel_acceptor 93862 52528 669517733 423931 0.06
Case novel_donor 93862 41334 669517733 318321 0.05
Control novel_acceptor 85458 47409 782827004 418368 0.05
Control novel_donor 85458 38049 782827004 317316 0.04
Expand details
  • # Novel junctions: total number of novel junctions for that particular cluster.
  • # Novel junc. in splice site: total number of novel junctions for that particular splice site in the cluster.
  • # Reads in cluster: total number of reads from the BAM files that constitutes the cluster.
  • # Novel junc. reads: reads associated to the novel junctions in the cluster and splice site.
  • Percent [%]: the percentage that the novel junction reads represent to the total number of reads in the cluster.
\[ \text{Percent [%]}=\frac{\text{# Novel junc. reads}}{\text{# Reads in cluster}}*100\% \]

7 Session info

Show/hide
## ─ Session info ───────────────────────────────────────────────────────────────────────────────────────────────────────
##  setting  value
##  version  R version 4.2.1 (2022-06-23)
##  os       Ubuntu 20.04.4 LTS
##  system   x86_64, linux-gnu
##  ui       X11
##  language (EN)
##  collate  en_US.UTF-8
##  ctype    en_US.UTF-8
##  tz       Etc/UTC
##  date     2023-06-05
##  pandoc   2.18 @ /usr/lib/rstudio-server/bin/quarto/bin/tools/ (via rmarkdown)
## 
## ─ Packages ───────────────────────────────────────────────────────────────────────────────────────────────────────────
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##  [2] /usr/local/lib/R/library
## 
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8 References

[1]
Garcı́a-Ruiz S, Zhang D, Gustavsson EK, Rocamora-Perez G, Grant-Peters M, Fairbrother-Browne A, et al. Splicing accuracy varies across human introns, tissues and age 2023. https://doi.org/10.1101/2023.03.29.534370.
[2]
Hummel M, Edelmann D, Kopp-Schneider A. Clustering of samples and variables with mixed-type data. PLOS ONE 2017;12:e0188274. https://doi.org/10.1371/journal.pone.0188274.